Chemiluminescent Western Blot Detection

As with other components in a western blotting system, there are many chemiluminescent western blotting substrate choices available. The appropriate selection of a substrate depends on the detection level (sensitivity) required, the target protein's abundance, the sample abundance and antibody availability. Use the tables below to find the right horseradish peroxidase (HRP) or alkaline phosphatase (AP) substrate for your experimental needs.

Find the right chemiluminescent substrate  Request sample

Recommended Chemiluminescent Substrates

 SuperSignal West AttoSuperSignal West Pico PlusSuperSignal West Dura
 SuperSignal West AttoSuperSignal West Pico PLUSSuperSignal West Dura
Choose when…Target is very low-abundant, sample is limited, or antibodies are limitedEveryday applications; offers improved sensitivity over base level ECLPerforming quantitative western blotting or maximum signal duration is needed
FeaturesHighest sensitivity Widest dynamic rangeLongest duration and linearity 
SensitivityLow femtogram to high attogramLow picogram to femtogramMid femtogram
Duration6 hoursup to 24 hours24 hours
Recommend antibody dilutions
(1 mg/ml stock)
1°: 1:5,000
2°: 1:100,000-1:250,000
1°: 1:1,000
2°: 1:20,000-1:100,000
1°: 1:5,000
2°: 1:50,000-1:250,000
Cat. No.A38554 (20 mL)
A38555 (100 mL)
A38556 (200 mL) 
34579 (20 mL)
34577 (200 mL)
34580 (500 mL)
34578 (1 L)
37071 (20 mL)
34075 (100 mL)
34076 (200 mL)
 Explore application dataExplore application dataExplore application data

See all available ECL substrates


Additional products for HRP substrates

Thermo Fisher Scientific productDescriptionCat. No.
SuperSignal Western Blot EnhancerMembrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it.46641
46640


Horseradish peroxidase (HRP) chemiluminescent substrates

HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting. Choice of the HRP chemiluminescent substrate used should be based on abundance of your target protein of interest, abundance of sample containing the target protein, quality of the antibodies and the level of sensitivity and type of instrumentation available for detection.
 

Featured webinar

Webinar: Detecting low abundant proteins in a western blot

In this webinar, we discuss specific methods from sample preparation through immunodetection that can help overcome challenges and improve signal to noise of low-abundance proteins for more successful western blot detection.

AP substrates

Novex AP Chemiluminescent Substrate

Novex AP Chemiluminescent Substrate
Long signal duration
SensitivityLow picogram
DurationOver 24 hours
Recommend antibody dilutions
(1 mg/ml stock)

1°: 1:500-5,000
2°: 1:2,000-1:10,000
When to useUsed standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes

Additional products for AP substrates

Thermo Fisher Scientific productDescriptionCat. No.
Novex AP Chemiluminescent Substrate Enhancer
(Nitro Block II)
20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranesWP20003
WesternBreeze Chromogenic Kit, anti-rabbitComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodiesWB7104
WesternBreezeChemiluminescent Kit, anti-rabbitComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodiesWB7106
WesternBreeze Chemiluminescent Kit, anti-goatComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodiesWB7108


Alkaline phosphatase (AP) chemiluminescent substrates

For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.  The emitted light is stable up to 24-96 hours allowing for multiple exposures.

Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.

Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.

Get started with our combo kits

SuperSignal West Substrate Combo Kit

SuperSignal West Substrate Combo Kit
Option A

Pico PLUS (500mL) + Trial size Atto (20mL)

Buy now

This combo kit includes:

  • SuperSignal West Pico PLUS Substrate (500mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)
     
Option B

Femto (100 mL) + Trial size Atto (20 mL)

Buy now

This combo kit includes:

  • SuperSignal West Femto Maximum Sensitivity Substrate (100mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)
Option C

Atto (100 mL) + Trial size Pico PLUS (20 m)

Buy now

This combo kit includes:

  • SuperSignal West Atto Ultimate Sensitivity Substrate (100mL)
  • SuperSignal West Pico PLUS Chemiluminescent Substrate trial size (20mL)
Option D

Pico PLUS (20 mL) and Atto (20 mL)

Buy now

This combo kit includes:

  • SuperSignal West Pico PLUS Chemiluminescent Substrate trial size (20mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)

Image capture and analysis for chemiluminescent western blots

Chemiluminescent western blot signal can be captured with X-ray film, charge-coupled device (CCD) camera–based digital imaging instruments, and phosphorimagers that detect chemiluminescence. X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, CCD camera–based imaging instruments offer the advantages of qualitative analysis, instant image capture and analysis, higher sensitivity, greater resolution and a larger dynamic range than film.

iBright Imaging System for chemiluminescent signal capture

iBright Imaging Systems

Capture and analyze publication quality images from chemiluminescent western blots with iBright Imaging Systems. These high-performance instruments enhance the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.

Explore our western blot documentation systems

Thermo Scientific CL-XPosure X-ray film for chemiluminescent documentation

X-Ray film

Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).

Recommended Chemiluminescent Substrates

 SuperSignal West AttoSuperSignal West Pico PlusSuperSignal West Dura
 SuperSignal West AttoSuperSignal West Pico PLUSSuperSignal West Dura
Choose when…Target is very low-abundant, sample is limited, or antibodies are limitedEveryday applications; offers improved sensitivity over base level ECLPerforming quantitative western blotting or maximum signal duration is needed
FeaturesHighest sensitivity Widest dynamic rangeLongest duration and linearity 
SensitivityLow femtogram to high attogramLow picogram to femtogramMid femtogram
Duration6 hoursup to 24 hours24 hours
Recommend antibody dilutions
(1 mg/ml stock)
1°: 1:5,000
2°: 1:100,000-1:250,000
1°: 1:1,000
2°: 1:20,000-1:100,000
1°: 1:5,000
2°: 1:50,000-1:250,000
Cat. No.A38554 (20 mL)
A38555 (100 mL)
A38556 (200 mL) 
34579 (20 mL)
34577 (200 mL)
34580 (500 mL)
34578 (1 L)
37071 (20 mL)
34075 (100 mL)
34076 (200 mL)
 Explore application dataExplore application dataExplore application data

See all available ECL substrates


Additional products for HRP substrates

Thermo Fisher Scientific productDescriptionCat. No.
SuperSignal Western Blot EnhancerMembrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it.46641
46640


Horseradish peroxidase (HRP) chemiluminescent substrates

HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting. Choice of the HRP chemiluminescent substrate used should be based on abundance of your target protein of interest, abundance of sample containing the target protein, quality of the antibodies and the level of sensitivity and type of instrumentation available for detection.
 

Featured webinar

Webinar: Detecting low abundant proteins in a western blot

In this webinar, we discuss specific methods from sample preparation through immunodetection that can help overcome challenges and improve signal to noise of low-abundance proteins for more successful western blot detection.

AP substrates

Novex AP Chemiluminescent Substrate

Novex AP Chemiluminescent Substrate
Long signal duration
SensitivityLow picogram
DurationOver 24 hours
Recommend antibody dilutions
(1 mg/ml stock)

1°: 1:500-5,000
2°: 1:2,000-1:10,000
When to useUsed standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes

Additional products for AP substrates

Thermo Fisher Scientific productDescriptionCat. No.
Novex AP Chemiluminescent Substrate Enhancer
(Nitro Block II)
20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranesWP20003
WesternBreeze Chromogenic Kit, anti-rabbitComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodiesWB7104
WesternBreezeChemiluminescent Kit, anti-rabbitComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodiesWB7106
WesternBreeze Chemiluminescent Kit, anti-goatComplete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodiesWB7108


Alkaline phosphatase (AP) chemiluminescent substrates

For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.  The emitted light is stable up to 24-96 hours allowing for multiple exposures.

Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.

Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.

Get started with our combo kits

SuperSignal West Substrate Combo Kit

SuperSignal West Substrate Combo Kit
Option A

Pico PLUS (500mL) + Trial size Atto (20mL)

Buy now

This combo kit includes:

  • SuperSignal West Pico PLUS Substrate (500mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)
     
Option B

Femto (100 mL) + Trial size Atto (20 mL)

Buy now

This combo kit includes:

  • SuperSignal West Femto Maximum Sensitivity Substrate (100mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)
Option C

Atto (100 mL) + Trial size Pico PLUS (20 m)

Buy now

This combo kit includes:

  • SuperSignal West Atto Ultimate Sensitivity Substrate (100mL)
  • SuperSignal West Pico PLUS Chemiluminescent Substrate trial size (20mL)
Option D

Pico PLUS (20 mL) and Atto (20 mL)

Buy now

This combo kit includes:

  • SuperSignal West Pico PLUS Chemiluminescent Substrate trial size (20mL)
  • SuperSignal West Atto Ultimate Sensitivity Substrate trial size (20mL)

Image capture and analysis for chemiluminescent western blots

Chemiluminescent western blot signal can be captured with X-ray film, charge-coupled device (CCD) camera–based digital imaging instruments, and phosphorimagers that detect chemiluminescence. X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, CCD camera–based imaging instruments offer the advantages of qualitative analysis, instant image capture and analysis, higher sensitivity, greater resolution and a larger dynamic range than film.

iBright Imaging System for chemiluminescent signal capture

iBright Imaging Systems

Capture and analyze publication quality images from chemiluminescent western blots with iBright Imaging Systems. These high-performance instruments enhance the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.

Explore our western blot documentation systems

Thermo Scientific CL-XPosure X-ray film for chemiluminescent documentation

X-Ray film

Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).

Chemiluminescent detection

Chemiluminescent detection occurs when energy from a chemical reaction is released in the form of light. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).

 HRP substrates (Horseradish peroxidase)AP substrates (Alkaline phosphatase)
Sensitivity

Femogram sensitivity

Picogram sensitivity
Signal generationImmediateGradually increases with signal maximum at ~30-60 minutes
Signal durationUp to 24 h24-96 hours
ConsiderationsCompatible with common buffers such as TBS and PBSNot compatible with phosphate buffers
When to useAntibodies or Probes conjugated to HRPAntibodies or Probes conjugated to AP


Find HRP linked secondary antibodies for chemiluminescent western blotting detection ›
Find AP conjugated secondary antibodies for chemiluminescent western blotting detection ›
 

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